Project Summary Increased levels of ambient particulate matter (PM) are consistently associated with cardiopulmonary and cardiovascular morbidity and mortality. Despite the apparent health risk of exposure, surprisingly little is known about the mechanisms underlying PM-mediated toxicity. PM emissions from a variety of combustion sources, such as organic wastes, diesel, gasoline, wood and cigarettes, contain persistent free radicals often termed environmentally persistent free radicals (EPFRs) due to their long half-lives in nature. EPFRs are a unique particle-pollutant system capable of redox-cycling in air and soils for days to weeks, resulting in the generation of reactive oxygen species. In our prior studies, we showed that inhalation of EPFRs decreased baseline cardiac function, but these effects were secondary to changes in pulmonary vascular resistance. The mechanism(s) underlying these vascular effects are as yet unknown; however, our preliminary data suggest that EPFR-mediated activation of the aryl hydrocarbon receptor (AhR) in pulmonary epithelial cells results in the release of vasoactive factors may play an important pathophysiological role. Our central hypothesis is that EPFR-mediated activation of AhR at the air-blood interface and mobilization of vasoactive mediators leads to activation/dysfunction of the pulmonary and systemic vasculature, resulting in cardiovascular disease initiation and progression. To test this hypothesis, Specific Aim 1 will elucidate the cellular mechanisms of vascular injury by testing whether EPFRs induce vascular dysfunction via activation of the AhR, Floxed mice deficient in epithelial AhR compared to control littermate mice will be chronically exposed to EPFRs, non-EPFR PM or filtered air by inhalation. Endothelium-dependent vascular reactivity, as well as markers for both endothelial dysfunction and activation will be assessed. Specific Aim 2 will identify a putative ligand promoting EPFR-induced AhR activation, and test whether this metabolite is associated with EPFR- mediated vascular dysfunction. This aim will use both target and untargeted mass spectrometry approaches to identify and characterize EFPR-induced lipid oxidation products that may serve as endogenous AhR agonists, so that we may correlate tissue and blood levels of these metabolites with vascular dysfunction. Completion of these aims will provide important new data linking EPFR-mediated oxidative stress, AhR activation, and cardiovascular disease. This information will be critical for assessing risks to those living in proximity to industrial or urban areas.